Reciprocally Retained Genes in the Angiosperm Lineage Show the Hallmarks of Dosage Balance

50 Pages·2017·5.23 MB·English

Plant Cell Advance Publication. Published on October 23, 2017, doi:10.1105/tpc.17.00313
LARGE-SCALE BIOLOGY ARTICLE
Reciprocally Retained Genes in the Angiosperm Lineage Show the Hallmarks
of Dosage Balance Sensitivity
Setareh Tasdighian1,2,3, Michiel Van Bel1,2,3, Zhen Li1,2,3, Yves Van de Peer1,2,3,4, Lorenzo
Carretero-Paulet1,2,3, Steven Maere1,2,3,*
1 Ghent University, Department of Plant Biotechnology and Bioinformatics, B-9052 Ghent,
Belgium
2 VIB Center for Plant Systems Biology, B-9052 Ghent, Belgium
3 Bioinformatics Institute Ghent, Ghent University, B-9052 Ghent, Belgium
4 Genomics Research Institute, University of Pretoria, Pretoria 0028, South Africa
*Corresponding author: [email protected]
Short title: Reciprocal retention and dosage balance sensitivity
One-sentence summary: Dosage balance sensitivity is the main reason why some genes are
preferentially retained after whole-genome multiplications but rarely duplicate through small-
scale duplication.
The author responsible for distribution of materials integral to the findings presented in this
article in accordance with the policy described in the Instructions for Authors
(www.plantcell.org) is: Steven Maere ([email protected]).
ABSTRACT
In several organisms, particular functional categories of genes, such as regulatory and complex-
forming genes, are preferentially retained after whole-genome multiplications but rarely duplicate
through small-scale duplication, a pattern referred to as reciprocal retention. This peculiar
duplication behavior is hypothesized to stem from constraints on the dosage balance between the
genes concerned and their interaction context. However, the evidence for a relationship between
reciprocal retention and dosage balance sensitivity remains fragmentary. Here, we identified
which gene families are most strongly reciprocally retained in the angiosperm lineage and studied
their functional and evolutionary characteristics. Reciprocally retained gene families exhibit
stronger sequence divergence constraints and lower rates of functional and expression divergence
than other gene families, suggesting that dosage balance sensitivity is a general characteristic of
reciprocally retained genes. Gene families functioning in regulatory and signaling processes were
much more strongly represented at the top of the reciprocal retention ranking than those
functioning in multiprotein complexes, suggesting that regulatory imbalances may lead to
stronger fitness effects than classical stoichiometric protein complex imbalances. Finally,
reciprocally retained duplicates are often subject to dosage balance constraints for prolonged
evolutionary times, which may have repercussions for the ease with which genome
multiplications can engender evolutionary innovation.
1 INTRODUCTION
2 Gene duplication is thought to be an important facilitator of evolutionary innovation and
3 phenotypic diversification, and hence the mechanisms governing the evolutionary fate of gene
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©2017 American Society of Plant Biologists. All Rights Reserved
4 duplicates have been studied intensively, see e.g. (Lynch and Conery, 2000; Lynch and Force,
5 2000; Wapinski et al., 2007; Conant and Wolfe, 2008). One class of duplications in particular,
6 whole-genome multiplications (WGM), has repeatedly been associated with increased
7 evolvability (Van de Peer et al., 2009a; Lohaus and Van de Peer, 2016; Soltis and Soltis, 2016).
8 Ancient WGMs have been documented in several evolutionary lineages such as vertebrates
9 (Christoffels et al., 2004; Jaillon et al., 2004; Dehal and Boore, 2005), ciliate protozoans (Aury et
10 al., 2006), hemiascomycetous yeasts (Wolfe and Shields, 1997; Wong et al., 2002; Kellis et al.,
11 2004) and especially flowering plants (Masterson, 1994; Soltis and Soltis, 1999; Otto and
12 Whitton, 2000; Cui et al., 2006; Van de Peer et al., 2009b; Van de Peer et al., 2009a; Vanneste et
13 al., 2014). Ancient WGM events have been inferred all over the angiosperm plant phylogeny,
14 including at the base of major clades such as the seed plants, angiosperms, core eudicots and
15 monocots (Jaillon et al., 2007; Jiao et al., 2011; Jiao et al., 2012; Amborella Genome Project,
16 2013; Jiao et al., 2014; Li et al., 2015). Additionally, more recent WGM events occurred
17 independently in many plant lineages (Van de Peer et al., 2009a; Vanneste et al., 2014; Soltis and
18 Soltis, 2016). Note however that despite the large number of ancient WGM events detectable in
19 present-day plant genomes, most newly formed plant polyploids still fail to establish themselves
20 for longer evolutionary timespans and do not diversify into successful new plant clades (Mayrose
21 et al., 2011; Mayrose et al., 2015). In this sense, WGM is thought to most often be an
22 evolutionary dead-end (Stebbins, 1950; Mayrose et al., 2011; Mayrose et al., 2015), but
23 occasionally an evolutionary success.
24 Although WGM events are generally followed by fractionation processes, removing most of the
25 duplicated genetic material from the genome (Freeling, 2009), some classes of genes have been
26 found to be preferentially retained after WGM. In Arabidopsis thaliana, transcriptional and
27 developmental regulators and signal transducers exhibit greater-than-average duplicate retention
28 after WGM (Blanc and Wolfe, 2004; Seoighe and Gehring, 2004; Maere et al., 2005; Freeling,
29 2009). Similar patterns were discovered in hemiascomycetous yeasts, e.g. for transcription factors
30 and ribosomal proteins (Papp et al., 2003; Conant and Wolfe, 2007), in the ciliate Paramecium
31 tetraurelia (Aury et al., 2006), in banana (Musa acuminata) (D'Hont et al., 2012) and in poplar
32 (Populus trichocarpa) (Carretero-Paulet and Fares, 2012; Rodgers-Melnick et al., 2012),
33 although for other species, the pattern is less clear (Carretero-Paulet and Fares, 2012).
34 Intriguingly, the classes of genes that were found to be preferentially retained after WGM also
35 exhibit preferential loss after small-scale duplication (SSD) (Blanc and Wolfe, 2004; Seoighe and
36 Gehring, 2004; Maere et al., 2005). This ‘reciprocal retention’ pattern has been hypothesized to
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37 originate from dosage balance effects (Maere et al., 2005; Freeling and Thomas, 2006; Birchler
38 and Veitia, 2007; Freeling, 2009; Birchler and Veitia, 2012). The dosage balance hypothesis
39 starts from the assumption that protein complexes, regulatory pathways or other complex systems
40 are often dosage balance-sensitive, i.e. that the stoichiometric balance of interacting components
41 in a system may affect the system’s function (Birchler et al., 2001; Veitia, 2002; Papp et al.,
42 2003; Veitia et al., 2008). (Some) components in such systems may therefore be subject to
43 dosage balance constraints that guarantee the proper functioning of the system. SSD of such
44 components would lead to a dosage imbalance and associated fitness defects and would therefore
45 be selected against. WGM, on the other hand, is expected to preserve the dosage balance of
46 molecular networks and multiprotein complexes. Moreover, dosage balance-sensitive genes
47 should preferentially be retained after WGM, as their deletion in a duplicated background is
48 expected to again lead to a dosage imbalance (Papp et al., 2003). In summary, the dosage balance
49 hypothesis predicts that dosage balance-sensitive genes should exhibit a strong reciprocal
50 retention pattern. However, the reverse is not necessarily true.
51 Several studies support the assertion that at least some ohnologs (WGM duplicates) are retained
52 because of dosage balance effects. Analyses on human ohnologs, remaining from the two rounds
53 of whole-genome duplication (WGD) at the base of the vertebrate lineage, have shown that these
54 ohnologs exhibit less copy-number variants (CNVs) on average than other genes (Makino and
55 McLysaght, 2010) and are more frequently associated with pathogenic CNVs (McLysaght et al.,
56 2014), suggesting that the function of some ohnologs is dosage-sensitive. Duplicates of metabolic
57 genes retained after the most recent WGD in the Arabidopsis thaliana lineage (the α event) were
58 found to cluster in the A. thaliana metabolic network, suggesting that their retention may serve to
59 maintain the relative dosage of genes functioning in the same metabolic pathway (Bekaert et al.,
60 2011). In Populus trichocarpa, approximately 55% of the duplicates remaining from the salicoid-
61 specific WGD were found to exhibit lower expression divergence than expected under a random
62 divergence model, and the genes concerned were also found to evolve under stronger purifying
63 selection on the sequence level than other genes, consistent with the predictions of the dosage
64 balance hypothesis (Rodgers-Melnick et al., 2012). However, it is largely unknown how these
65 dosage balance sensitivity hallmarks relate to the reciprocal retention strength of the genes
66 concerned, given that many WGM duplicates are likely not reciprocally retained, and those that
67 are may not necessarily be the same ones as those that are dosage balance-sensitive.
68 A study in the Glycine genus (Coate et al., 2016) found that genes of Gene Ontology (GO)
69 classes and metabolic pathways that exhibit strong reciprocal retention characteristics after the
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70 shared WGD in the Glycine lineage (5-13 million years ago [Mya]) exhibit less variable gene
71 expression within and between species and less variable expression fold-changes upon additional
72 more recent polyploidization events (~0.5 Mya) than other gene classes, and links these
73 observations to dosage balance-sensitivity. However, not all genes of a broad functional class of
74 genes that is known to be more prone to reciprocal retention, e.g. all transcription factors, are
75 necessarily strongly reciprocally retained, and there might be strongly reciprocally retained genes
76 in other gene classes as well. A next step towards assessing the extent to which reciprocal
77 retention patterns can be explained by dosage balance effects is to identify which specific gene
78 families, irrespective of class, exhibit the strongest reciprocal retention pattern, and to study their
79 functional and evolutionary characteristics in the light of predictions made by the dosage balance
80 theory. One of these predictions is that reciprocally retained duplicates, if dosage balance-
81 sensitive, should exhibit less functional divergence and expression divergence than other
82 duplicates, at least until the dosage balance constraints can be circumvented (Casneuf et al., 2006;
83 Veitia et al., 2008; Coate et al., 2011; Conant et al., 2014). Another prediction is that the protein
84 products of reciprocally retained genes should interact with other proteins or DNA/RNA, and that
85 disturbances in the dosage balance of such interactions, e.g. by overexpressing or
86 underexpressing the genes concerned or changing the number of gene family copies, should have
87 effects on an organism’s phenotype and fitness (Veitia et al., 2008).
88 Because of the large number of retained genome duplications, plants are ideally suited to study
89 the differential impact of WGM and SSD on the duplication dynamics of individual gene
90 families. Here, we modeled the dynamics of gene family size evolution over a phylogeny of 37
91 angiosperm species using a stochastic birth-death model, taking into account both discrete WGM
92 events and continuous SSD. In all, we analyzed 9,178 core angiosperm gene families using our
93 model and ranked those families in terms of their inferred reciprocal retention strength across the
94 angiosperm clade. Gene families associated with processes that are thought to be more prone to
95 dosage balance effects, such as transcriptional regulation, signal transduction and development,
96 were found to generally exhibit stronger reciprocal retention patterns, confirming previous
97 studies. In addition, gene families with stronger reciprocal retention patterns were found to
98 exhibit reduced nonsynonymous sequence divergence, functional divergence and expression
99 divergence, in accordance with the predictions of the dosage balance hypothesis. A literature
100 study for the top-11 reciprocally retained gene families, ten of which have been experimentally
101 characterized to at least some extent, revealed that most of these gene families feature
102 overexpression/deletion phenotypes consistent with dosage balance sensitivity, and that at least
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103 some of them interact directly or indirectly with members of other gene families that are also
104 highly reciprocally retained. Together, our results show that dosage balance sensitivity is a major
105 factor determining whether or not genes duplicate preferentially through WGM.
106 RESULTS
107
108 Identification of gene families with strong reciprocal retention constraints
109 Modeling gene family size evolution after WGM and SSD
110 To identify gene families that preferentially duplicate through WGM in the angiosperm lineage,
111 we fitted a stochastic gene birth-death (BD) model to the size (gene count) profiles of individual
112 gene families in 37 sequenced angiosperms (see Methods). Given an input species tree and the
113 associated gene counts for a particular gene family, our model computes a maximum likelihood
114 estimate for a single parameter λ that represents both the SSD birth rate of new duplicates in the
115 gene family concerned and the gene loss rate (where the loss rate captures the loss of genes
116 produced through WGM and SSD as well as the loss of ancestrally present genes). Reciprocally
117 retained gene families should have both a low SSD birth rate and a low WGM loss rate, and
118 hence a low λ (see also Methods). Gene families showing a perfect reciprocal retention pattern
119 (no SSD duplicates, SSD loss hence irrelevant, and no loss after WGMs) should have an inferred
120 λ value of 0 under our model.
121 We based our BD model on the gene family size evolution model of Hahn et al. (2005) but
122 extended it to account for WGM events, as in Rabier et al. (2014) (see Methods). Rabier et al.
123 (2014) found that their gene count-based BD model compared favorably to an alternative method
124 based on gene tree-species tree reconciliation, even though less information is used (only gene
125 counts, no sequences). Gene count-based models have the added benefit that they are
126 computationally less complex than reconciliation models (Rabier et al., 2014), which facilitated
127 running the model on thousands of individual gene families, rather than on combined sets of gene
128 families, as done previously (Hahn et al., 2005; Rabier et al., 2014; Tiley et al., 2016).
129 We applied the model to previously published gene count data for 9,178 core gene families
130 across 37 angiosperm species (Figure 1B, Supplemental Data Set 1) (Li et al., 2016). The
131 minimum λ value attained across all gene families was 0.354, showing that none of the gene
132 families has a perfect reciprocal retention pattern. The distribution of optimal λ values for all
133 9,178 gene families is shown in Figure 1A, and the λ estimates for all gene families are given in
134 Supplemental Data Set 1. The average λ value inferred across gene families is 0.827. Since time
135 in our analyses is measured in terms of the average number of substitutions per codon (see
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136 Methods), this result suggests that gene duplication/loss rates are on average on the same order of
137 magnitude as substitution rates, which fits earlier observations (see e.g. (Lynch and Conery,
138 2000)). For some of the analyses below, we classified the gene families into groups by putting
139 group cutoffs one standard deviation above and below the data mean, i.e. at λ = 0.601 and
140 λ = 1.053, respectively. The 1,077 gene families with an optimal λ below 0.601, which exhibit
141 the strongest reciprocal retention pattern, are hereafter referred to as ‘top’ gene families.
142 Similarly, the 965 families with an optimal λ above 1.053, which exhibit the gene count patterns
143 that are least consistent with preferential retention after WGM and low SSD duplicability, are
144 hereafter referred to as ‘bottom’ gene families. It is important to mention that these bottom gene
145 families are not necessarily gene families that have high SSD duplicate counts (see also further).
146 Robustness of the inferred gene family ranking to gene count errors, WGM misplacement and
147 species subsampling
148 BD models such as the one used here have previously been shown to be fairly robust to errors in
149 gene family counts, e.g. due to incomplete genome assemblies (Han et al., 2013). To test the
150 robustness of our λ ranking to gene count errors, we partitioned the gene families into groups
151 with λ estimates in intervals of 0.1 in size and perturbed the gene counts of 100 randomly
152 sampled families per λ interval (sampling with replacement) by adding or subtracting, for every
153 species, a number of genes sampled from a Gaussian distribution N(µ,σ) with mean µ = 0 and a
154 given standard deviation σ (numbers are rounded to the nearest integer). We recalculated the
155 maximum-likelihood λ estimates for all modified gene family count profiles under the model
156 outlined in the previous section and compared these to the original estimates. When sampling
157 errors from N(0,0.5), corresponding to a change in ~4% of the gene counts on average (for
158 profiles containing 37 species, this translates to gene count changes of magnitude 1 for 1 to 2
159 species on average and very occasionally a gene count change of magnitude 2), the average new
160 λ estimate remains very close to the original λ estimate for every λ bin, and the standard
161 deviation of the new λ estimates in every bin is small enough not to cause major changes in the
162 rank-ordering of gene families (Supplemental Figure 1).
163 If we increase the error by sampling from N(0,0.65), corresponding to a change in ~12% of the
164 gene counts on average, or gene count changes of magnitude 1 for 4 to 5 species on average and
165 occasionally a gene count change of magnitude 2, the average new λ estimate is slightly higher
166 than the original λ estimate for every λ bin, and the standard deviation of the new λ estimates in
167 the bins increase, but the original rank-order is still largely conserved.
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168 When further increasing the error by sampling from N(0,0.80) and N(0,1), corresponding on
169 average to a change in ~21% and ~34% of the gene counts, respectively, and increasingly more
170 gene count changes of magnitude 2 or more, the average new λ estimates for each bin become
171 progressively higher, particularly for lower λ bins. Moreover, the associated standard deviations
172 increase to the extent that the original λ rank-order is severely influenced, although the overall
173 increasing λ trend is still visible, particularly for gene families with low original λ values. In
174 other words, the most strongly reciprocally retained gene families still rank highly when adding a
175 substantial amount of gene count noise. These results show that the BD model is robust to small
176 gene count errors, but is increasingly sensitive to larger errors, as expected.
177 An alternative source of error in the inferred gene family ranking could be the misplacement of
178 WGMs on the species tree or the inclusion of erroneously inferred WGMs. Although most of the
179 WGM events that have been inferred across the angiosperm tree are supported by multiple types
180 of evidence across several studies, the status and exact timing of some of the more recently
181 proposed WGMs, particularly in the monocot clade (Tang et al., 2010; D'Hont et al., 2012; Jiao et
182 al., 2014), is less clear-cut and still being consolidated (Ming et al., 2015; Wang et al., 2015;
183 McKain et al., 2016; Tiley et al., 2016). We assessed the robustness of the gene family ranking to
184 WGM inference uncertainties by running simulations for seven alternatives to the default WGM
185 scenario presented in Figure 1B (Supplemental Table 1). The λ-based gene family rankings were
186 found to be highly correlated across all WGM scenarios tested (Spearman rank correlation 0.996
187 to 1.0, Supplemental Table 1), showing that WGM uncertainties in a few lineages do not lead to
188 drastically different inferences of which gene families exhibit the strongest or weakest reciprocal
189 retention patterns.
190 We also used a subsampling approach to investigate how robust the inferred λ values are to
191 changes in the number of species used in the model and their taxonomic sampling profile. For
192 each of a selected subset of gene families (the 37 gene families with ranks 1, 2...10, 11, 21,...91,
193 101, 201...901, 1001, 2001...9001 in the λ-based ranking), we removed the gene count of one
194 species at random from the gene family profile and re-estimated λ for the reduced data set. This
195 was done 37 times for each gene family in the subset, and the average and standard deviation of
196 the resulting λ estimates were recorded (see Supplemental Data Set 2). The results indicate that,
197 when using 36 plant species instead of 37 for λ inference, the average λ values and the λ-based
198 ranking across the selected gene family subset are overall very similar to the values and ranking
199 inferred from the complete dataset (Spearman rank correlation 0.995), indicating that omitting
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200 one species from the dataset has only a minor impact on the resulting gene family ranking. A few
201 gene families, however, exhibit leave-one-out λ standard deviations that are greater than others,
202 and these are generally also the gene families that exhibit sizeable shifts in their average leave-
203 one-out λ value (see Supplemental Data Set 2). Note that such leave-one-out λ standard
204 deviations cannot be used to infer reliable jackknife confidence intervals on the inferred λ values
205 for the complete dataset, as the gene counts of the 37 species are not independent and identically
206 distributed (iid) as required. The gene counts are not independent because of their evolutionary
207 linkage and have different distributions by virtue of differences in their WGM history and
208 evolutionary distances from the gene family root. The same problem precludes the use of
209 bootstrap and cross-validation techniques to estimate λ confidence intervals.
210 We also performed a similar subsampling analysis removing the gene counts for five species at
211 random instead of one (see Supplemental Data Set 2). While the resulting leave-five-out
212 λ standard deviations are generally higher than in the leave-one-out case, the gene family ranking
213 based on the average leave-five-out λ values is still overall very similar to the original ranking in
214 the gene family subset investigated (Spearman rank correlation 0.976), indicating that the λ-
215 based gene family ranking is robust to substantial changes in the species tree composition used
216 for their inference. Interestingly, the leave-five-out λ averages (and to a much lesser extent the
217 leave-one-out λ averages) are systematically greater than the original λ values estimated on the
218 full data set (with most differences in the range of 0.01-0.05, see Supplemental Data Set 2). The
219 reason for this is not entirely clear but may have something to do with the fact that the inference
220 of λ values is progressively less constrained in datasets with fewer species and gene counts,
221 thereby widening the λ probability distributions and shifting their modes asymmetrically to the
222 right (as λ values below 0 are impossible). It is important to note, however, that the true λ values
223 are not important in the present context, only their ranking is.
224 Top gene families have more duplicates retained in syntenic blocks and fewer tandem duplicates
225 than bottom gene families
226 To independently assess whether the BD model truly identifies ‘top’ gene families that primarily
227 duplicate through WGM and have low SSD duplicability, we looked at the fraction of duplicates
228 retained in syntenic blocks remnant from WGMs (hereafter referred to as block duplicates) and
229 the fraction of tandem duplicates in each gene family across species, using a custom-built
230 PLAZA database (Proost et al., 2015) incorporating the 37 plant species studied here (see
231 Methods). No syntenic blocks were recovered for barley (Hordeum vulgare), likely because of
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232 the highly fragmented nature of its genome sequence. This species was therefore omitted from
233 the analysis below. When all remaining species were considered together, top families showed a
234 significantly higher fraction of block duplicates on average (0.839 ± 0.154) than bottom families
235 (0.442 ± 0.298) (one-sided Mann-Whitney U test, Bonferroni-corrected p = 1.68e-161) (Figure
236 2). Reciprocally, bottom families showed a significantly higher fraction of tandem duplicates
237 (0.432 ± 0.268) than top families (0.092 ± 0.102) (one-sided Mann-Whitney U test, Bonferroni-
238 corrected p = 5.93e-167). When species were considered individually, differences in the fractions
239 of block and tandem duplicates among top versus bottom families were significant for all species
240 except physic nut (Jatropha curcas), birdsfoot trefoil (Lotus japonicus), date palm (Phoenix
241 dactylifera) and Populus trichocarpa (Supplemental Figure 2). Interestingly, when looking at all
242 species combined (Figure 2), the bias in the distribution of block and tandem percentages across
243 top gene families is much stronger than across bottom gene families. Top gene families tend to
244 have very high block duplicate percentages and very low tandem duplicate percentages, while
245 there is less bias towards high block or high tandem duplicate percentages for bottom families.
246 Overall, these results indicate that the top gene families identified by the BD model are likely to
247 be gene families with a strong reciprocal retention pattern. The size evolution of bottom gene
248 families, on the other hand, is generally affected by both WGM and SSD duplication and loss
249 events, or in other words, these gene families are the least subject to reciprocal retention
250 constraints.
251 It is also evident from the species-specific K distributions for duplicate pairs in the top and
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252 bottom gene families that the top gene families are enriched in WGM-derived duplicates
253 (Supplemental Figure 3). Vanneste et al. (2014) previously reported K ranges for the most recent
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254 WGMs in 24 of the species studied here. For all of those species except H. vulgare, the K
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255 distribution of duplicates in the top gene families exhibits a clear peak in the relevant K range.
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256 Furthermore, the top gene family K distributions for eudicot species that did not undergo any
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257 additional WGM after the γ triplication shared by all core eudicots, such as grape (Vitis vinifera),
258 papaya (Carica papaya), cacao (Theobroma cacao), castor bean (Ricinus communis), wild
259 strawberry (Fragaria vesca), watermelon (Citrullus lanatus) and the Cucumis and Prunus spp.,
260 display a prominent peak in the K range 1-3 that is likely associated with this γ triplication.
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261 Similarly, the top gene family K distributions for the cereal grasses rice (Oryza sativa), purple
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262 false brome (Brachypodium distachyon), sorghum (Sorghum bicolor) and foxtail millet (Setaria
263 italica) display, next to a prominent peak in the K range of the most recent shared ρ whole-
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264 genome duplication (WGD) (Paterson et al., 2004), a secondary peak at higher K values that
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265 likely corresponds to the older σ and/or τ WGDs inferred previously in the Poales and monocot
266 lineages, respectively (Tang et al., 2010; Jiao et al., 2014; McKain et al., 2016).
267 A combined ranking based on both the λ value and the block duplicate percentage of gene
268 families
269 Although the top gene families with low λ generally show a significant enrichment for block
270 duplicates derived from WGM events, a few of the gene families high up in the λ-based ranking
271 exhibit low block duplicate percentages and high tandem duplicate percentages (see
272 Supplemental Data Set 1), suggesting that the BD model-based ranking of gene families
273 according to reciprocal retention strength is not perfect. Indeed, some gene families might exhibit
274 a gene count pattern similar to the patterns expected for reciprocally retained gene families
275 because of stochastic birth-death effects rather than reciprocal retention. To filter out erroneously
276 highly ranked gene families from the top of the λ-based list, we used the overall block duplicate
277 percentage of the gene families (i.e. the number of block duplicates in a gene family across all
278 species, divided by the total number of genes in the gene family) as an independent source of
279 evidence for reciprocal retention. In addition to the λ-based ranking, gene families were ranked
280 according to decreasing block duplicate percentage, and both rankings were merged into a
281 combined ranked list of reciprocally retained gene families by averaging the constituent ranks
282 (Supplemental Data Set 1). The λ-based and combined rankings exhibit a Spearman rank
283 correlation of 0.887. The functional and evolutionary analyses described in the next sections were
284 performed on both the λ-based and combined rankings, leading to very similar results, although
285 the differences between top and bottom gene families in the combined ranking are generally a bit
286 more outspoken. The results on the combined ranking are reported in the main text and figures,
287 whereas the results on the λ-only ranking can be found in the Supplemental Data. For the
288 analyses on the combined ranking reported in the main text, the new top and bottom gene family
289 categories were defined as the 1000 highest- and lowest-ranked gene families, respectively.
290 Li et al. (2016) previously analyzed the same gene count dataset analyzed here to study the
291 duplication characteristics of gene families across the angiosperms and broadly classified the
292 gene families in three groups with different duplication behaviors, namely 5,097 ‘single-copy’
293 gene families, in which the genes exhibit a strong preference to return to the single-copy state
294 after SSD or WGM, 1,249 ‘multi-copy’ gene families, which have multiple retained duplicates in
295 most species, and 2,832 ‘intermediate’ gene families, in which duplicates are generally retained
10